Promiscuity in the geometry of electrostatic interactions between the Escherichia coli multidrug resistance transporter MdfA and cationic substrates
Promiscuity in the geometry of electrostatic interactions between the Escherichia coli multidrug resistance transporter MdfA and cationic substrates
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DOI：
10.1074/jbc.m412332200
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发表时间：
2005-01-28
影响因子：
4.8
通讯作者：
Bibi, E
中科院分区：
文献类型：
--
作者：
Adler, J;Bibi, E
大肠杆菌多药转运蛋白MdfA含有单个膜包埋的带电残基（Glu-26），其在阳离子底物的识别中起关键作用（埃德加，M和Bibi，E.（1999）EMBO J. 18，822-832）。使用失活突变体（MdfA-E26 T），我们分离出一个自发的第二位点突变（MdfA-E26 T/V335 E），该突变重新建立了转运蛋白对阳离子药物的识别。只有在位置335处的负电荷能够恢复失活突变体MdfA-E26 T的功能。有趣的是，这两个遗传相互作用的残基位于沿着MdfA二级结构沿着的遥远且不同的区域。Glu-26位于跨膜螺旋1的周质半部分，如图所示，位置335处的互补电荷位于连接跨膜螺旋10和11的细胞质环内。通过交联研究了两个残基之间的空间关系。构建缺乏半胱氨酸的功能性分裂形式的MdfA，并在位置26和335处引入半胱氨酸对。引人注目的是，结果表明残基26和335在空间上相邻，表明它们都构成MdfA的多药物识别口袋的一部分。即使关键的酸性残基被放置在口袋的另一面上，静电相互作用也与阳离子底物保持不变，这一事实揭示了多药物识别和运输中混杂的另一个维度。
The Escherichia coli multidrug transporter MdfA contains a single membrane-embedded charged residue (Glu-26) that plays a critical role in the recognition of cationic substrates (Edgar, M, and Bibi, E. (1999) EMBO J. 18, 822-832). Using an inactive mutant (MdfA-E26T), we isolated a spontaneous second-site mutation (MdfA-E26T/V335E) that re-established the recognition of cationic drugs by the transporter. Only a negative charge at position 335 was able to restore the functioning of the inactive mutant MdfA-E26T. Intriguingly, the two genetically interacting residues are located at remote and distinct regions along the secondary structure of MdfA. Glu-26 is located in the periplasmic half of transmembrane helix 1, and as shown here, the complementing charge at position 335 resides within the cytoplasmic loop connecting transmembrane helices 10 and 11. The spatial relation between the two residues was investigated by cross-linking. A functional split version of MdfA devoid of cysteines was constructed and introduced with a cysteine pair at positions 26 and 335. Strikingly, the results indicate that residues 26 and 335 are spatially adjacent, suggesting that they both constitute parts of the multidrug recognition pocket of MdfA. The fact that electrostatic interactions are preserved with cationic substrates even if the critical acidic residue is placed on another face of the pocket reveals an additional dimension of promiscuity in multidrug recognition and transport.